Presentation on theme: "Lek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia"— Presentation transcript:
1 Lek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia STRUCTURAL PROPERTIES OF ANTIMICROBIAL PEPTIDES ACTING ON BACTERIAL MEMBRANESBoštjan JapeljLek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia
2 Antibiotics – “miracle drugs” Bacterial resistance is becoming a major problem in modern medicine
3 Cationic antimicrobial peptides: - up to 50 aminoacids longa net positive charge of at least +2 (Arg, Lys)- antimicrobial activity against G-, G+ bacteria, fungi, protozoa, viruses,anticancer activity, effectors of innate immune response- 4 structural classes:ref:
4 - act on membranes and intracellular targets, ref.: Matsuzaki, K.,. Biochim Biophys Acta, (1-2): p-advantage: fast acting, resistance is unlikely to develop, able to neutralize bacterial endotoxins and prevent development of sepsis
5 C12LF11 lauryl-FQWQRNIRKVR-NH2 P3-55 octanoyl-FWRIRIRR – NH2 LACTOFERRINLF FQWQRNIRKVR-NH2C12LF lauryl-FQWQRNIRKVR-NH2P octanoyl-FWRIRIRR – NH2Membrane models:LPS (lipopolysaccharide): model for baterial membraneSDS (sodium dodecyl sulphate): model for bacterial membraneDPC (dodecyl phosphocholine): model for eucaryotic membrane
6 NMR study of LF11 + S-LPS, LF11 + SDS, LF11 + DPC LF11 + S-LPS: trNOECPMG-T2 experiment:+LF11LPSkoffLF11-LPSTRNOE between aromatic and aliphatic side chains in 2 mM LF11 upon addition of 1/20 of molar ratio of LPS (b) and LTA (c). The reference NOESY spectrum of LF11 is shown in (a). Spectra were recorded at a mixing time of 150 ms.
7 LF11 + S-LPS Family of 3D structures of LF11 in complex with LPS. ( basic, hydrophobic,Complex between LF11 and LPS441 Å2 of surface area buriedpolar residues)
8 Binding motif: LF11 :Phe1 , Arg5 , Lys9 , Arg11 Comparison of LPS interaction motifs in FhuA (left)1, LF11 (center)2 and polymyxin B (right)3,4 in the same orientation with respect to LPS, which would be in front of the plane of the pageBinding motif:LF11 :Phe1 , Arg5 , Lys9 , Arg11FhuA :Phe355, Lys439 , Arg384 , Lys351PmxB :Phe6 , Dab8,9 , Dab3 , Dab11 Ferguson, A. D., Hofmann, E., Coulton, J. W., Diederichs, K., and Welte, W. (1998) Science 282: 2215–22202Japelj, B., Pristovšek, P., Majerle, A., Jerala, R. J Biol Chem, (17): p3Pristovšek, P. and J. Kidrič, J Med Chem, (22):4Pristovšek, P., Simčič, S., Wraber, B., Urleb, U. , J Med Chem, :
9 Comparison of structures LF11+S-LPS, LF11+SDS, LF11+DPC
10 N-terminal part of LF11 is protected from fluoresscence quenching Fluorescence quenchingLF11 FQWQRNIRKVR-NH2F0, F…Fluorescence emission intensity in theabsence and presence of the quencher(Q)[Q]… concentration of the quencherKSV… Stern-Volmer quenching constant
11 -acylation enhances antimicrobial activity against G- and G+ bacteria C12LF11:-acylation enhances antimicrobial activity against G- and G+ bacteria-acylation stabilizes secondary structureCD spectra of LF11 and C12LF11 inDPC micellesFamily of structures of C12LF11 in DPC11Japelj, B., Zorko, M., Majerle, A., Pristovšek, P.,et al.. JACS, (2007), 129:
12 OCTANOYL-F W R I R I R R – NH2 P3-55:OCTANOYL-F W R I R I R R – NH2
17 Normalized I/Iref ratios of HN-Ha cross peaks in NOESY spectrum P3-55 in SDSP3-55 in DPC
18 Molecular dynamics of P3-55 in DPC Total energy (left) and temperature (right) of the system (P DPC SOL + 4 Cl-) during first 2 ns of simulationDPCDPC + P3-55time [ps]*Ratios between princpal moments of inertia of DPC during simulation of P3-55 in DPC. Principal moments of inertia are shown in the table.*moments of inertia in units 104 amu nm2. Asymetry parameter, a, defined as a = (2I1-I2-I3)/(I1+I2+I3)Order parameter tensor elementsof DPC micelle for the simulationof DPC micelle a) and DPC micelle + P3-55 b). –SCD=2/3Sxx + 1/2SyyRadial density of P3-55 in complex with DPC micelle.DPC coordinates were taken fromTieleman, D.P., et al. J. Phys Chem. B. 2000, 104:
19 Outer membraneCytoplasmic membraneMechanism of interaction of ANEPID peptides with the membrane of Gram-negative bacteria.
20 Acknowledgements:Andreja Majerle, Primož Pristovšek, Mateja Zorko, Roman Jerala (NIC, Ljubljana)Miha Kotnik, Katja Kristan, Drago Kuzman, Andrej Preželj, Jan Humljan, Petra Igličar, Vjekoslava Car, Uroš Urleb (Lek, Drug Discovery, Ljubljana)co-workers from the EU project ANEPID (Antimicrobial Endotoxin-neutralazingPeptides to Combat Infectious Deseases):Dagmar Zweytick, Karl Lohner (Graz)Guillermo Martinez de Tejada, Ignacio Moriyon, Susana Sanchez-Gomez (Pamplona)Sylvie E. Blondelle (San Diego, CA)Klaus Brandenburg, Jörg Andrä (Borstel)
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